Method for inhibition of pathogenic microorganisms
a technology for pathogenic microorganisms and inhibitors, which is applied in the direction of peptide sources, applications, genetic material ingredients, etc., can solve the problems of few reports of primary human macrophage transfection with dna plasmids, insufficient yield of recombinant methods, etc., and achieve the effect of inhibiting preventing the growth of a microorganism
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example 1
[0081] The following example describes the optimization of transfection efficiency for transfection of mRNA into macrophages using enhanced green fluorescent protein (eGFP).
[0082] Monocytes were isolated from human whole blood by centrifugation through Ficoll-Hypaque. The mononuclear cell layer was washed in RPMI 1640+saline, and the monocytes counted. Approximately 1×106 monocytes were dispensed into the wells of 24 well plates (Falcon, Becton Dickinson), and allowed to adhere for one hour. The monolayers were then washed three times to remove non-adherent cells. The resulting cell monolayers consisted of M. tuberculosis (Erdman). Cells were placed at 100,000 / well into 8 well chambered coverslips (Nalge-Nunc intl., Naperville, Ill.) and allowed to adhere for 2 hours in RPMI1640 including penicillin (0.05 units / ml), streptomycin (0.05 μg / ml), L-glutamine, and 10% autologous human serum. Non-adherent cells were then removed with 3 washes with warm PBS, and the medium replaced with a...
example 2
[0090] The following example demonstrates the relative toxicities of mRNAs encoding GFP vs hBD-2.
[0091] Cationic lipids are known to be toxic to mammalian cells at high concentration (Freedland et al., Biochem Mol Med 59:144-53, 1996), as are defensins (Lichtenstein et al., Blood 68:1407-1410, 1986). The present inventors therefore sought to determine the maximum dose of GFP mRNA / oligofectin G complex which could be applied to the macrophages, and whether human β-defensin (hBD-2) mRNA had greater toxicity. Specifically, the inventors determined the ability of human monocyte-derived macrophages (MDM) to reduce MTT 24 hours after treatment with increasing concentrations of either GFP mRNA (produced as described in Example 1) or hBD-2 mRNA (see below) complexed with Oligofectin G.
[0092] hBD-2 cDNA was produced by RT-PCR using human tracheal epithelial cell mRNA as a template and published primer sequences (Harder et al., Nature 387:861, 1997, incorporated herein by reference in its e...
example 3
[0094] The following example demonstrates the production of hBD-2, and association with intracellular M. tuberculosis following mRNA transfection.
[0095] It was thought that the ability of hBD-2 to affect the growth of M. tuberculosis within macrophages would depend in part on the ability of the defensin protein to gain access to the mycobacteria. Therefore, immunocytochemistry was performed using a specific rabbit anti-human hBD-2 antiserum to determine if hBD-2 protein was produced following transfection of macrophages with hBD-2 mRNA, and to determine where in the macrophages the protein localized.
[0096] For immunocytochemistry, cells were grown on eight chamber slides, fixed in formalin at 4° C., and washed in 1M glycine. Immunohistochemistry was carried out as described (Yount et al., J Biol Chem 274:26249-58, 1999), using specific hBD-2 antibody (a gift of T. Ganz) or non-immune serum, and visualized using the Vector ABC kit (Vectorlabs). Acid-fast staining of mycobacteria wa...
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